Software for the display of chromatographic separation data

ABSTRACT

Techniques and systems for displaying chromatographic data using a graphical user interface are provided. Chromatographic separation data that represent a series of measurements for multiple samples detected at a single point along the length of a channel as they move through the channel past that point can be displayed on a display device of a computer system as a series of bands, the bands being arranged to resemble output from an electrophoresis gel. The bands may be aligned using a marker included in each sample.

This application is a divisional of U.S. Application Ser. No.12/761,787, filed on Apr. 16, 2010,which is a continuation of U.S.Application Ser. No. 11/811,031, filed on June 7, 2007, and issued onApr. 20, 2010, as U.S. Pat. No. 7,702,470,which is a continuation ofU.S. Application Ser. No. 10/900,761, filed on Jul. 27, 2004, and issuedon Sep. 16, 2008, as U.S. Pat. No. 7,426,442, which is a continuation ofU.S. Application Ser. No. 10/443,657, filed on May 22, 2003, and issuedon Dec. 21, 2004, as U.S. Pat. No. 6,834,240, which is a continuation ofU.S. Application Ser. No. 10/155,324, filed on May 24, 2002, and issuedon Aug. 26, 2003, as U.S. Pat. No. 6,611,768, which is a continuation ofU.S. Application Ser. No. 09/223,070, filed on Dec. 29, 1998, and issuedon Aug. 6, 2002 as U.S. Pat. No. 6,430,512, all of which claim thebenefit of U.S. Provisional Application No. 60/068,980, filed on Dec.30, 1997. The above documents are hereby incorporated by reference.

BACKGROUND OF THE INVENTION

The present invention relates to the graphical display of data. Morespecifically, the invention relates to the display of chromatographicseparation data that are a series of measurements over time in agraphical format, e.g., as a series of bands.

Analysis of biological samples often requires the resolution andcharacterization of the constituent elements of the sample. The moreinteresting of these constituents are macromolecular structures, e.g.,proteins, nucleic acids, carbohydrates, and the like. Typically,analytical separation of macromolecular species is carried out usingchromatographic techniques. Of particular widespread use areelectrophoretic techniques that employ slab-gels disposed between twoglass plates as a separation matrix. Samples containing themacromolecular species that are sought to be analyzed, are introducedinto wells at one end of the slab gel. An electric current is thenapplied through the gel drawing the macromolecular species through thegel by virtue of a charge either on, or otherwise associated with themacromolecular species. Each sample travels through the gelsubstantially linearly, e.g., in a lane corresponding to its well.

As the sample progresses through the gel, molecules of different sizeand/or charge will have different mobilities through the gel, and willseparate into bands that reflect their relative size and/or charge. Uponcompletion, the gel is stained or otherwise examined whereby the variousbands can be visualized and compared with standard macromolecularcompounds, e.g., having standard molecular weight and/or charge, e.g.,isolectric point.

For example, in the case of protein analysis using sodium dodecylsulfatepolyacrylamide gel electrophoresis (SDS-PAGE), proteins are drawnthrough the gel matrix in a highly charged detergent micelle (SDS) toensure that the proteins, regardless of charge, will electrophoresethrough the gel. The proteins will travel at a rate that is proportionalto their size. Once separated, the protein bands are stained, e.g., withcoomassie blue or silver staining, to permit analysis and recordation,e.g., as a photograph or a digital or analog scan.

Similarly, nucleic acid analyses utilize a similar gel system, e.g.,agarose or polyacrylamide gel. Upon application of a current through thegel, the nucleic acid samples, again disposed in wells at one end(anode) of the gel, will electrophorese through the gel. The polymer gelpresents a sieving matrix, where larger nucleic acid fragments thatotherwise having the same charge:mass ratio as smaller fragments, willtravel more slowly through the gel than the smaller fragments. Uponcompletion of electrophoresis, the lanes of samples are analyzed for thepattern of the bands (or “ladder” as it is often termed). Analysis ofthe bands may be carried out by adding a fluorescent intercalating agentto the gel, or by incorporating a radioactive label within the nucleicacid fragments, followed by contacting the gel with a photographic film.

Typically, electrophoresis gels run multiple samples within the sameslab gel along with one or more standards or markers, which are used tocharacterize the sample constituents. For example, in size-basedseparations, standards typically have a range of known molecularweights. Sample constituents are then compared to the standards todetermine their molecular weights, e.g., by interpolation. Suchstandards must generally be run in the same gel as the sample, in orderto provide assurances that the standard was subjected to the sameseparation conditions, e.g., gel composition, electric current,temperature, or other parameters affecting separations.

Despite the efficacy of these slab gel electrophoresis, however, suchmethods are quickly being supplanted by automated procedures thatgenerate a stream of digital data. This data, in its raw form, mayexhibit the non-linearities described earlier, or different ones, ornone at all. Such data may be generated, for example, by passing asample in front of a sensor. Alternatively, it is also possible todigitize the raw information presented in a traditional gel by scanningit to produce a series of measurements. The display of such informationis not provided by current systems.

What is therefore needed are techniques for displaying chromatographicseparation data that are a series of measurements over time in a formatsimilar to that of traditional gel presentations. Moreover, it would bebeneficial to provide normalization of such data, if desired.

SUMMARY OF THE INVENTION

The present invention provides innovative techniques for displaying aseries of measurements, e.g., as acquired from a microfluidic capillaryseparation experiment, in a gel-like format. This gel-like formatdisplays chromatographically separated and detected species as bands ofvarying width and intensity in a vertical lane format, e.g., as aladder. This format further permits the side-by-side display ofchromatographic data from multiple different samples, which data can benormalized to internal standards. In particular, chromatographic dataobtained in the form of optical intensity, e.g., fluorescence, UVabsorbance, or the like, as a function of time, e.g., as a chromatogram,can be displayed in a band format, as a ladder. Further, seriallyacquired data from analysis of multiple samples, e.g., from serialseparations in the same separation system, as opposed to parallelacquired data, e.g., from a multi-lane slab gel, can be displayedside-by-side, and can be normalized to one or more standards.

In one embodiment, the invention provides a computer implemented methodof displaying chromatographic separation data. A series of measurementsindicating presence of constituents in a sample at a scanning locationover time is received. The series of measurements for the sample isdisplayed as a series of bands. Additionally, peaks in the series ofmeasurements can be identified that correspond to one or more markers.The series of measurements can be scaled so that any displayed bandsthat correspond to the one or more markers are aligned withpredetermined locations or markers from a previous or the same sample.

In another embodiment, the invention provides a computer implementedmethod of displaying chromatographic separation data. A series ofmeasurements indicating the presence of constituents and at least onemarker in a first sample at a scanning location over time is received. Aseries of measurements indicating the presence of constituents and atleast one marker in a second sample at a scanning location over time isalso received. The series of measurements for the first sample isdisplayed as a series of bands. The series of measurements for the firstsample is analyzed to identify at least one peak that corresponds to theat least one marker. Similarly, the series of measurements for thesecond sample is analyzed to identify at least one peak that correspondsto the at least one marker. The series of measurements for the secondsample are scaled so that the displayed bands that correspond to the atleast one marker in the first and second samples are aligned whendisplayed. Lastly, the series of measurements for the second sample isdisplayed as a series of bands adjacent to the bands for the firstsample.

In another embodiment, the invention provides a computer implementedmethod of graphically presenting chromatographic separation data.Chromatographic data for a sample is acquired, the chromatographic datafor the sample including a set of constituents and a set of markers. Aposition of each marker in the chromatographic data is determined inorder to define a range of positions. Additionally, an intensity of eachmarker in the chromatographic data is determined in order to define arange of intensities. The position of each constituent in thechromatographic data is determined by scaling the position to the rangeof positions and the intensity of each constituent in thechromatographic data is determined by scaling the position to the rangeof range of intensities. The position and intensity of each constituentin the chromatographic data is then presented in a graphical format.

A particularly useful application of these methods and processes is inthe field of capillary electrophoresis. In capillary electrophoresis,materials to be separated based upon their size, e.g., nucleic acids,proteins, etc., are introduced into one end of a narrow bore capillarychannel, which typically includes a separation matrix, e.g., a polymersolution or gel, disposed therein. Application of an electric fieldthrough the capillary channel then draws the sample through the channel.The presence of the polymer solution or gel, or alternatively,differential molecular charges of the macromolecular species, imparts adifferent mobility to the different macromolecular species in thesample, depending upon their size. Because a single thin channel is usedfor a given separation, typically only a single sample can be analyzedat any time, but channels could be utilized in parallel. However, asingle capillary channel can serially analyze multiple sampleseffectively and this obviates the need for separately run ranges ofstandards. Instead, internal standards, e.g., of known molecular weight,typically are included with the sample materials, to provide a referencepoint against which the sample constituents or components may becompared. Typically, such standards will fall outside of the expectedseparation range for the sample constituents, e.g., have much larger orsmaller molecular weights then the sample constituents. This permits thestandards to be readily identified as the standards, and prevents themfrom interfering with the analysis of the sample constituents.Alternatively, differential labeling techniques may be used, whereby thestandards may be distinguished from other constituents of the samplematerial by virtue of their incorporating a distinguishable label, e.g.,having different light absorbing or emitting properties.

Separated species are generally detected at a single point along thelength of the capillary channel as they move past that point. Typically,detection is carried out through the incorporation or association of adetectable labeling group with the various macromolecular species. Thedata from the detector is typically displayed as peaks of opticalintensity as a function of time, e.g., as a chromatogram, for eachsample analyzed. Analysis of additional samples is then carried outserially, e.g., one after another, in the same capillary system, givingrise to multiple separate plots of optical intensity peaks vs. time.Because these data are obtained from separate runs, with potentiallyvarying conditions, these multiple plots make it very difficult tocompare data from different samples.

In one aspect of the present invention, data obtained in the form of atypical chromatographic plot of intensity peaks are displayed as aseries of bands of varying widths and intensities, in a verticalladder-like format. Further, a user may toggle back and forth betweenthe different display modes, e.g., chromatogram and gel-like displays,as well as manipulate of the data to permit optimal comparison andanalysis of this data, e.g., normalization of data to standards,interpolation/extrapolation of data to characterize data from thedifferent samples and different constituents of each sample.

A further understanding of the nature and advantages of the inventiondescribed herein may be realized by reference to the remaining portionsof the specification and the attached drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows an embodiment of a microfluidic device.

FIG. 2. shows a system including a microfluidic instrument and acomputer system.

FIG. 3 illustrates an example of a computer system that may be utilizedto execute the software of an embodiment of the invention.

FIG. 4 illustrates a system block diagram of the computer system of FIG.3.

FIG. 5 shows a flowchart of a process of displaying chromatographicseparation data that is a series of measurements at a scanning locationover time as a series of bands.

FIG. 6 shows a screen display of an embodiment of the inventionincluding a series of bands.

FIG. 7 shows a flowchart of a process of normalizing chromatographicseparation data in which the samples include one or more markers.

FIG. 8A and 8B show screen displays that illustrate the normalizingprocess of series of bands.

FIG. 9 shows a flowchart of another process of normalizingchromatographic separation data in which the samples include one or moremarkers.

FIGS. 10A-10E show screen displays of embodiments of the invention.

FIG. 11 shows a flowchart of a process of displaying chromatographicseparation data for multiple samples.

FIG. 12 illustrates in further detail a flowchart of a preferred processof generating a graphical display of chromatographic data for onesample.

FIG. 13 depicts a gel display window according to one embodiment of thepresent invention.

FIG. 14 depicts a gel display window according to another embodiment ofthe present invention.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS Introduction

The present invention relates to the display of data from a chemicalassay. More particularly, the present invention provides techniques fordisplaying microfluidic capillary separation data in a “gel” format onthe display device of a computer. First, the data generated by theexperiment is loaded into the computer. This data can include, amongother information, data representing fluorescence levels observed in thesample being analyzed typically as a function of retention time in anelectrophoretic separation system, e.g. capillary electrophoresis. Thesefluorescence levels typically represent one or more standards and one ormore constituents of samples. The present invention displays the data asa series of bands like a ladder, in a manner substantially similar to atraditional gel.

In one embodiment, the present invention creates a normalization curveusing a set of standards (or markers) of known characteristics, e.g.,molecular weight. The constituents of the samples are displayed as aseries of bands (also called a “ladder”). These bands (i.e., thefluorescence levels) may be displayed as a positive (white bands on ablack background), a negative (black bands on a white background), orusing one of a variety of color combinations. The fluorescence data maybe displayed in normalized and unnormalized formats. The unknown sampleladder(s) are normalized to the standard ladder by matching thestandards embedded in each sample ladder to those of the standardladder.

One aspect of the present invention is the conversion of seriallygenerated data into a more conventional parallel format. Data generatedby systems such as the exemplary system described herein are in a serialformat, and would normally be expected to be displayed as such. However,by converting this information into a gel display, the display ofchromatographic data by the present invention is made simpler, lessexpensive (on a per-run basis), and more repeatable than conventionalgel assays.

Graphical Display of Chromatographic Data

As noted above, the techniques described herein are particularly usefulin analyzing data from capillary electrophoresis applications. However,it will be appreciated that these methods and processes also are usefulin a wide variety of chromatographic separation systems, e.g.,conventional column chromatography, HPLC, FPLC, mass spectrometry,scanned slab gel methods, and the like.

As also noted, the methods and processes are useful in capillaryelectrophoretic systems that serially analyze multiple samples within asingle capillary channel. In particularly preferred aspects, a planarmicrofluidic device that includes multiple sample reservoirs coupled toa single separation channel is used in conjunction with the dataanalysis and presentation methods and processes described herein.Examples of such systems are described in detail in copending, commonlyassigned PCT Publication WO 98/49548, and incorporated herein byreference. In particular, multiple different samples disposed inseparate sample wells in the body of the device, are separately injectedinto a single separation channel within the device, one after another.

Exemplary Microfluidic Devices

In preferred aspects, certain of the devices, methods and systemsdescribed herein which are used to produce the chromatographicseparation data described herein, employ electrokinetic materialtransport systems, and preferably, controlled electrokinetic materialtransport systems. As used herein, “electrokinetic material transportsystems” include systems which transport and direct materials within aninterconnected channel and/or chamber containing structure, through theapplication of electrical fields to the materials, thereby causingmaterial movement through and among the channel and/or chambers, i.e.,cations will move toward the negative electrode, while anions will movetoward the positive electrode.

Such electrokinetic material transport and direction systems includethose systems that rely upon the electrophoretic mobility of chargedspecies within the electric field applied to the structure. Such systemsare more particularly referred to as electrophoretic material transportsystems. Other electrokinetic material direction and transport systemsrely upon the electroosmotic flow of fluid and material within a channelor chamber structure, which results from the application of an electricfield across such structures. In brief, when a fluid is placed into achannel which has a surface bearing charged functional groups, e.g.,hydroxyl groups in etched glass channels or glass microcapillaries,those groups can ionize. In the case of hydroxyl functional groups, thisionization, e.g., at neutral pH, results in the release of protons fromthe surface and into the fluid, creating a concentration of protons atnear the fluid/surface interface, or a positively charged sheathsurrounding the bulk fluid in the channel. Application of a voltagegradient across the length of the channel, will cause the proton sheathto move in the direction of the voltage drop, i.e., toward the negativeelectrode.

“Controlled electrokinetic material transport and direction,” as usedherein, refers to electrokinetic systems as described above, whichemploy active control of the voltages applied at multiple, i.e., morethan two, electrodes. Rephrased, such controlled electrokinetic systemsconcomitantly regulate voltage gradients applied across at least twointersecting channels. Controlled electrokinetic material transport isdescribed in Published PCT Application No. WO 96/04547, to Ramsey, whichis incorporated herein by reference in its entirety for all purposes.

FIG. 1 shows one embodiment of a microfluidic device that can be usedwith the invention. A device 1 includes multiple wells that areinterconnected with microchannels or fluid conduits. As shown, device 1includes 16 wells in which four wells are slightly larger than the othernine wells. Sample wells 3 can hold fluid samples and buffer wells 5 canbe utilized to hold buffer solutions to aid the microfluidic separationprocess. For example, in macromolecular separation applications, e.g.,nucleic acid and protein separations, the buffer solution can include apolymer that sieves the macromolecular species by size as they aredriven through it by means of electrophoresis, similar to using agaroseor polyacrylamide gels. The samples and buffer solutions can include anintercalating dye that becomes more fluorescent upon binding to themacromolecular species. Each sample is electrokinetically moved from itswell to a central separating channel 7. A small amount of the sample isinjected into and electrophoresed in separating channel 7, where theconstituents and markers in the sample separate by size and pass a laser(e.g., red laser at 635 nm) that excites the fluorescent dye bound tothe macromolecular species. After excitation, the portion of the samplethat has reached a scanning location is scanned to produce a series ofmeasurements of fluorescent intensity vs. time. Although fluorescentlabels will be described herein, other types of label including lightabsorbing labels, radioactive labels, and the like can be utilized withthe invention.

Typically, the samples in sample wells 3 are serially driven throughseparating channel 7. Buffer wells 5 can be utilized to “wash” theseparating channel between samples. A graphical representation 21 of thedevice is shown. The graphical representation can be displayed for auser and includes the wells of the device without the microchannels. Thewells are shown with a letter identification for the rows and a numberidentification for the columns. Accordingly, each well (and the sampleor buffer therein) can be identified by a combination of letters andnumbers (e.g., “A3”).

In general, a microfluidic device can include two intersecting channelsor fluid conduits, e.g., interconnected, enclosed chambers, and threeunintersected termini. The intersection of two channels refers to apoint at which two or more channels are in fluid communication with eachother, and encompasses “T” intersections, cross intersections, “wagonwheel” intersections of multiple channels, or any other channel geometrywhere two or more channels are in such fluid communication. Anunintersected terminus of a channel is a point at which a channelterminates not as a result of that channel's intersection with anotherchannel, e.g., a “T” intersection.

In preferred aspects, the devices will include at least threeintersecting channels having at least four unintersected termini. In abasic cross channel structure, where a single horizontal channel isintersected and crossed by a single vertical channel, controlledelectrokinetic material transport operates to controllably directmaterial flow through the intersection, by providing constraining flowsfrom the other channels at the intersection. For example, assuming onewas desirous of transporting a first material through the horizontalchannel, e.g., from left to right, across the intersection with thevertical channel. Simple electrokinetic material flow of this materialacross the intersection could be accomplished by applying a voltagegradient across the length of the horizontal channel, i.e., applying afirst voltage to the left terminus of this channel, and a second, lowervoltage to the right terminus of this channel, or by allowing the rightterminus to float (applying no voltage). However, this type of materialflow through the intersection would result in a substantial amount ofdiffusion at the intersection, resulting from both the natural diffusiveproperties of the material being transported in the medium used, as wellas convective effects at the intersection.

In controlled electrokinetic material transport, the material beingtransported across the intersection is constrained by low level flowfrom the side channels, e.g., the top and bottom channels. This isaccomplished by applying a slight voltage gradient along the path ofmaterial flow, e.g., from the top or bottom termini of the verticalchannel, toward the right terminus. The result is a “pinching” of thematerial flow at the intersection, which prevents the diffusion of thematerial into the vertical channel. The pinched volume of material atthe intersection may then be injected into the vertical channel byapplying a voltage gradient across the length of the vertical channel,i.e., from the top terminus to the bottom terminus. In order to avoidany bleeding over of material from the horizontal channel during thisinjection, a low level of flow is directed back into the side channels,resulting in a “pull back” of the material from the intersection.

In addition to pinched injection schemes, controlled electrokineticmaterial transport is readily utilized to create virtual valves thatinclude no mechanical or moving parts. Specifically, with reference tothe cross intersection described above, flow of material from onechannel segment to another, e.g., the left arm to the right arm of thehorizontal channel, can be efficiently regulated, stopped andreinitiated, by a controlled flow from the vertical channel, e.g., fromthe bottom arm to the top arm of the vertical channel. Specifically, inthe “Off” mode, the material is transported from the left arm, throughthe intersection and into the top arm by applying a voltage gradientacross the left and top termini. A constraining flow is directed fromthe bottom arm to the top arm by applying a similar voltage gradientalong this path (from the bottom terminus to the top terminus). Meteredamounts of material are then dispensed from the left arm into the rightarm of the horizontal channel by switching the applied voltage gradientfrom left to top, to left to right. The amount of time and the voltagegradient applied dictates the amount of material that will be dispensedin this manner.

Although described for the purposes of illustration with respect to afour way, cross intersection, these controlled electrokinetic materialtransport systems can be readily adapted for more complex interconnectedchannel networks, e.g., arrays of interconnected parallel channels.

As used herein, the term “microscale” or “microfabricated” generallyrefers to structural elements or features of a device which have atleast one fabricated dimension in the range of from about 0.1 μm toabout 500 μm. Thus, a device referred to as being microfabricated ormicroscale will include at least one structural element or featurehaving such a dimension. When used to describe a fluidic element, suchas a passage, chamber or conduit, the terms “microscale,”“microfabricated” or “microfluidic” generally refer to one or more fluidpassages, chambers or conduits which have at least one internalcross-sectional dimension, e.g., depth, width, length, diameter, etc.,that is substantially within the given dimensions.

In the devices of some embodiments of the present invention, themicroscale channels or chambers preferably have at least onecross-sectional dimension are also within the given dimensions.Accordingly, the microfluidic devices or systems prepared in accordancewith the present invention typically include at least one microscalechannel, usually at least two intersecting microscale channels, andoften, three or more intersecting channels disposed within a single bodystructure. Channel intersections may exist in a number of formats,including cross intersections, “T” intersections, or any number of otherstructures whereby two channels are in fluid communication.

The body structure of the microfluidic devices described hereintypically comprises an aggregation of two or more separate layers whichwhen appropriately mated or joined together, form the microfluidicdevice of the invention, e.g., containing the channels and/or chambersdescribed herein. Typically, the microfluidic devices described hereinwill comprise a top portion, a bottom portion, and an interior portion,wherein the interior portion substantially defines the channels andchambers of the device.

A variety of substrate materials may be employed as the bottom portion.Typically, because the devices are microfabricated, substrate materialswill be selected based upon their compatibility with knownmicrofabrication techniques, e.g., photolithography, wet chemicaletching, laser ablation, air abrasion techniques, injection molding,embossing, and other techniques. The substrate materials are alsogenerally selected for their compatibility with the full range ofconditions to which the microfluidic devices may be exposed, includingextremes of pH, temperature, salt concentration, and application ofelectric fields. Accordingly, in some preferred aspects, the substratematerial may include materials normally associated with thesemiconductor industry in which such microfabrication techniques areregularly employed, including, e.g., silica based substrates, such asglass, quartz, silicon or polysilicon, as well as other substratematerials, such as gallium arsenide and the like. In the case ofsemiconductive materials, it will often be desirable to provide aninsulating coating or layer, e.g., silicon oxide, over the substratematerial, and particularly in those applications where electric fieldsare to be applied to the device or its contents.

In additional preferred aspects, the substrate materials will comprisepolymeric materials, e.g., plastics, such as polymethylmethacrylate(PMMA), polycarbonate, polytetrafluoroethylene (TEFLON™),polyvinylchloride (PVC), polydimethylsiloxane (PDMS), polysulfone, andthe like. Such polymeric substrates are readily manufactured usingavailable microfabrication techniques, as described above, or frommicrofabricated masters, using well known molding techniques, such asinjection molding, embossing or stamping, or by polymerizing thepolymeric precursor material within the mold (see U.S. Pat. No.5,512,131). Such polymeric substrate materials are preferred for theirease of manufacture, low cost and disposability, as well as theirgeneral inertness to most extreme reaction conditions. Again, thesepolymeric materials may include treated surfaces, e.g., derivatized orcoated surfaces, to enhance their utility in the microfluidic system,e.g., provide enhanced fluid direction, e.g., as described in PCTPublication WO 98/46438, and which is incorporated herein by referencein its entirety for all purposes.

In many embodiments, the microfluidic devices will include an opticaldetection window disposed across one or more channels and/or chambers ofthe device. Optical detection windows are typically transparent suchthat they are capable of transmitting an optical signal from thechannel/chamber over which they are disposed. Optical detection windowsmay merely be a region of a transparent cover layer, e.g., where thecover layer is glass or quartz, or a transparent polymer material, e.g.,PMMA, polycarbonate, etc. Alternatively, where opaque substrates areused in manufacturing the devices, transparent detection windowsfabricated from the above materials may be separately manufactured intothe device.

These devices may be used in a variety of applications, including, e.g.,the performance of high throughput screening assays in drug discovery,immunoassays, diagnostics, genetic analysis, and the like. As such, thedevices described herein, will often include multiple sampleintroduction ports or reservoirs, for the parallel or serialintroduction and analysis of multiple samples. Alternatively, thesedevices may be coupled to a sample introduction port, e.g., a pipetor,which serially introduces multiple samples into the device for analysis.Examples of such sample introduction systems are described in, e.g., PCTPublications WO 98/00231 and WO 98/00705, each of which is herebyincorporated by reference in its entirety for all purposes.

Instrumentation

The systems described herein generally include microfluidic devices, asdescribed above, in conjunction with additional instrumentation forcontrolling fluid or material transport and direction within thedevices, detection instrumentation for detecting or sensing results ofthe operations performed by the system, processors, e.g., computers, forinstructing the controlling instrumentation in accordance withpreprogrammed instructions, receiving data from the detectioninstrumentation, and for analyzing, storing and interpreting the data,and providing the data and interpretations in a readily accessiblereporting format.

FIG. 2 shows an embodiment of a microfluidic instrument that can beutilized with the invention. A microfluidic instrument 51 includes acover 53. The cover overlies a chamber in which a microfluidic device isplaced. Preferably, the microfluidic device is configured so that it canonly be placed in the correct orientation (e.g., by a notch in onecorner of the device). After the microfluidic device is placed in thechamber of microfluidic instrument 51, the user lowers cover 53. In oneembodiment, the cover includes multiple electrodes (not shown) that areplaced in the wells of the microfluidic device when the cover islowered. The electrodes are used to drive the fluids through themicrochannels of the microfluidic device. In a preferred embodiment,each electrode is separately powered.

Microfluidic instrument 51 is shown electronically connected to acomputer system 71 by a cable 73 (e.g., a serial cable). Computer system71 can be utilized to control microfluidic instrument 51 and analyze theresulting data. Additionally, the electronics to control themicrofluidic station can be included in the instrument.

Once chromatographic separation data is obtained, computer system 71 canbe utilized to analyze and display the data. Although the computersystem is shown connected to the microfluidic instrument directly, thecomputer system need not be directly connected to the instrument orindeed even at the same location. For example, the computer system canbe at a remote site for analysis and receive the chromatographicseparation data through a network (e.g., the Internet) or a portablestorage medium (e.g., floppy drive). Accordingly, the invention is notlimited to the specific configurations shown.

A variety of controlling instrumentation may be utilized in conjunctionwith the microfluidic devices described above, for controlling thetransport and direction of fluids and/or materials within the devices ofthe present invention. For example, in many cases, fluid transport anddirection may be controlled in whole or in part, using pressure basedflow systems that incorporate external or internal pressure sources todrive fluid flow. Internal sources include microfabricated pumps, e.g.,diaphragm pumps, thermal pumps, lamb wave pumps and the like that havebeen described in the art. See, e.g., U.S. Pat. Nos. 5,271,724,5,277,556, and 5,375,979 and Published PCT Application Nos. WO 94/05414and WO 97/02357. In such systems, fluid direction is often accomplishedthrough the incorporation of microfabricated valves, which restrictfluid flow in a controllable manner. See, e.g., U.S. Pat. No. 5,171,132.

As noted above, the systems described herein preferably utilizeelectrokinetic material direction and transport systems. As such, thecontroller systems for use in conjunction with the microfluidic devicestypically include an electrical power supply and circuitry fordelivering appropriate voltages to a plurality of electrodes that areplaced in electrical contact with the fluids contained within themicrofluidic devices. Examples of particularly preferred electricalcontrollers include those described in, e.g., U.S. patent applicationSer. No. 08/888,064, and PCT Publication WO 98/00707, the disclosures ofwhich are hereby incorporated herein by reference in their entirety forall purposes. In brief, the controller uses electric current control inthe microfluidic system.

The electrical current flow at a given electrode is directly related tothe ionic flow along the channel(s) connecting the reservoir in whichthe electrode is placed. This is in contrast to the requirement ofdetermining voltages at various nodes along the channel in a voltagecontrol system. Thus the voltages at the electrodes of the microfluidicsystem are set responsive to the electric currents flowing through thevarious electrodes of the system. This current control is lesssusceptible to dimensional variations in the process of creating themicrofluidic system in the device itself. Current control permits fareasier operations for pumping, valving, dispensing, mixing andconcentrating subject materials and buffer fluids in a complexmicrofluidic system. Current control is also preferred for moderatingundesired temperature effects within the channels.

In the microfluidic systems described herein, a variety of detectionmethods and systems may be employed, depending upon the specificoperation that is being performed by the system. Often, a microfluidicsystem will employ multiple different detection systems for monitoringthe output of the system. Examples of detection systems include opticalsensors, temperature sensors, pressure sensors, pH sensors, conductivitysensors, and the like. Each of these types of sensors is readilyincorporated into the microfluidic systems described herein. In thesesystems, such detectors are placed either within or adjacent to themicrofluidic device or one or more channels, chambers or conduits of thedevice, such that the detector is within sensory communication with thedevice, channel, or chamber. The phrase “within sensory communication”of a particular region or element, as used herein, generally refers tothe placement of the detector in a position such that the detector iscapable of detecting the property of the microfluidic device, a portionof the microfluidic device, or the contents of a portion of themicrofluidic device, for which that detector was intended. For example,a pH sensor placed in sensory communication with a micro scale channelis capable of determining the pH of a fluid disposed in that channel.Similarly, a temperature sensor placed in sensory communication with thebody of a microfluidic device is capable of determining the temperatureof the device itself.

Particularly preferred detection systems include optical detectionsystems for detecting an optical property of a material within thechannels and/or chambers of the microfluidic devices that areincorporated into the microfluidic systems described herein. Suchoptical detection systems are typically placed adjacent a microscalechannel of a microfluidic device, and are in sensory communication withthe channel via an optical detection window that is disposed across thechannel or chamber of the device. Optical detection systems includesystems that are capable of measuring the light emitted from materialwithin the channel, the transmissivity or absorbance of the material, aswell as the materials spectral characteristics. In preferred aspects,the detector measures an amount of light emitted from the material, suchas a fluorescent or chemiluminescent material. For example, in thepresent invention, such detectors may include laser fluorescence devicesthat detect fluorescence induced by exposure to laser radiation togenerate the chromatographic data thus displayed. This is a preferredembodiment used in the present invention.

As such, the detection system will typically include collection opticsfor gathering a light based signal transmitted through the detectionwindow, and transmitting that signal to an appropriate light detector.Microscope objectives of varying power, field diameter, and focal lengthmay be readily utilized as at least a portion of this optical train. Thelight detectors may be photodiodes, avalanche photodiodes,photomultiplier tubes, diode arrays, or in some cases, imaging systems,such as charged coupled devices (CCDs) and the like. In preferredaspects, photodiodes are utilized, at least in part, as the lightdetectors. The detection system is typically coupled to the computer(described in greater detail below), via an AD/DA converter, fortransmitting detected light data to the computer for analysis, storageand data manipulation.

In the case of fluorescent materials, the detector will typicallyinclude a light source that produces light at an appropriate wavelengthfor activating the fluorescent material, as well as optics for directingthe light source through the detection window to the material containedin the channel or chamber. The light source may be any number of lightsources that provides the appropriate wavelength, including lasers,laser diodes and LEDs. Other light sources may be required for otherdetection systems. For example, broad band light sources are typicallyused in light scattering/transmissivity detection schemes, and the like.Typically, light selection parameters are well known to those of skillin the art.

The detector may exist as a separate unit, but is preferably integratedwith the controller system, into a single instrument. Integration ofthese functions into a single unit facilitates connection of theseinstruments with a computer system (described below), by permitting theuse of few or a single communication port(s) for transmittinginformation between the controller, the detector and the computer.

Computer System

As noted above, either or both of the controller system and/or thedetection system can be coupled to an appropriately programmed processoror computer which functions to instruct the operation of theseinstruments in accordance with preprogrammed or user input instructions,receive data and information from these instruments, and interpret,manipulate and report this information to the user. As such, thecomputer is typically appropriately coupled to one or both of theseinstruments (e.g., including an AD/DA converter as needed).

FIG. 3 illustrates an example of a computer system that may be used toexecute the software of an embodiment of the invention. FIG. 3 shows acomputer system 71 that includes a display 73, screen 75, cabinet 77,keyboard 79, and mouse 81. Mouse 81 may have one or more buttons forinteracting with a graphical user interface. Cabinet 77 houses a CD-ROMdrive 83, system memory and a hard drive (see FIG. 4) which may beutilized to store and retrieve software programs incorporating computercode that implements the invention, data for use with the invention, andthe like. Although CD-ROM 85 is shown as an exemplary computer readablestorage medium, other computer readable storage media including floppydisk, tape, flash memory, system memory, and hard drive may be utilized.Additionally, a data signal embodied in a carrier wave (e.g., in anetwork including the Internet) may be the computer readable storagemedium.

FIG. 4 shows a system block diagram of computer system 71 used toexecute the software of an embodiment of the invention. As in FIG. 3,computer system 71 includes monitor 73 and keyboard 79, and mouse 81.Computer system 71 further includes subsystems such as a centralprocessor 91, system memory 93, fixed storage 95 (e.g., hard drive),removable storage 97 (e.g., CD-ROM drive), display adapter 99, soundcard 101, speakers 103, and network interface 105. Other computersystems suitable for use with the invention may include additional orfewer subsystems. For example, another computer system could includemore than one processor 91 (i.e., a multi-processor system) or a cachememory.

The system bus architecture of computer system 71 is represented byarrows 107. However, these arrows are illustrative of anyinterconnection scheme serving to link the subsystems. For example, alocal bus could be utilized to connect the central processor to thesystem memory and display adapter. Computer system 71 shown in FIG. 4 isbut an example of a computer system suitable for use with the invention.Other computer architectures having different configurations ofsubsystems may also be utilized.

The computer system typically includes appropriate software forreceiving user instructions, either in the form of user input into a setparameter fields, e.g., in a GUI, or in the form of preprogrammedinstructions, e.g., preprogrammed for a variety of different specificoperations. The software then converts these instructions to appropriatelanguage for instructing the operation of the fluid direction andtransport controller to carry out the desired operation. The computerthen receives the data from the one or more sensors/detectors includedwithin the system, and interprets the data, either provides it in a userunderstood format, or uses that data to initiate further controllerinstructions, in accordance with the programming, e.g., such as inmonitoring and control of flow rates, temperatures, applied voltages,and the like. Thus, a graphical display of chromatographic separationdata according to the present invention provides greater flexibility inthe display of such data, and features heretofore unseen in the displayof such information.

Device Integration

Although the devices and systems specifically illustrated herein aregenerally described in terms of the performance of a few or oneparticular operation, it will be readily appreciated from thisdisclosure that the flexibility of these systems permits easyintegration of additional operations into these devices. For example,the devices and systems described will optionally include structures,reagents and systems for performing virtually any number of operationsboth upstream and downstream from the operations specifically describedherein. Such upstream operations include sample handling and preparationoperations, e.g., cell separation, extraction, purification,amplification, cellular activation, labeling reactions, dilution,aliquoting, and the like. Similarly, downstream operations may includesimilar operations, including, e.g., separation of sample components,labeling of components, assays and detection operations. Assay anddetection operations include without limitation, probe interrogationassays, e.g., nucleic acid hybridization assays utilizing individualprobes, free or tethered within the channels or chambers of the deviceand/or probe arrays having large numbers of different, discretelypositioned probes, receptor/ligand assays, immunoassays, and the like.

Display of Chromatographic Separation Data

The chromatographic separation data can be analyzed on a computer systemthat is connected to the microfluidic instrument or one that receivesthe data remotely. The chromatographic separation data typically is inthe form of a measured intensity (be it fluorescence or otherwise) at ascanning location vs. time. A graphical plot of intensity vs. time canbe very useful, but many scientists and researchers are not accustomedto this format for electrophoresis separation analysis. Further, theside by side comparison of such data from multiple samples can bedifficult.

FIG. 5 shows a high level flowchart of a process of displayingchromatographic data that is a series of measurements at a scanninglocation over time as a series of bands. At a step 151, the computersystem receives a series of measurements at a scanning location overtime. The series of measurements can be fluorescent intensities thatwere measured at the scanning location of the microfluidic device as asample was electrokinetically pulled through the separation channel. Thecomputer system displays the series of measurements as a series of bandsat a step 153. The series of bands can resemble a conventionalelectrophoresis gel that users may find more familiar. A graphical plotof intensity vs. time can also be displayed.

FIG. 6 shows a screen display of an embodiment of the invention. Awindow 161 includes a graphical representation 163 of a microfluidicdevice (see FIG. 1). A circle 165 indicates the sample well that iscurrently selected or being processed. The graphical representation canalso include other information including an identification number forthe microfluidic device, the date and time the microfluidic device wasread, and the like.

A window area 167 can show graphical plots of intensity vs. time foreach of the sample wells that have been processed. Each plot isidentified by the letter and number combination that uniquely identifiesthe row and column of the sample well (e.g., “A1” in this case). Agraphical plot 169 shows the measured fluorescent intensity vs. time forthe sample well identified as A1. The sample in well A1 is a ladder of amacromolecule, which in this example is a DNA ladder. If a sampledesignated by a user to include a ladder, the graphical plot isidentified as a “Ladder” as shown, otherwise, the graphical plots areidentified as “Sample.”

A window area 171 includes a series of bands 173. The series of bandswas generated from the series of measurements at a scanning locationover time that produced graphical plot 169. However, series of bands 173resembles the output from a conventional electrophoresis gel. As will bediscussed in more detail below, window 161 includes many otherinnovative features.

In preferred embodiments, the samples (and ladders) include markers ofknown characteristics (e.g., molecular weight). The markers can belabeled with a distinctive marker such as fluorescent labels of adifferent wavelength or color so that they can be distinguished fromconstituents of the sample or they can be identified by other means(e.g., markers that are lighter or heavier than the expectedconstituents of a sample can be readily identified). The markers can beutilized to normalize the display of series of measurements as follows.

FIG. 7 shows a flowchart of a process of normalizing chromatographicseparation data in which the samples include one or more markers.Although the steps of the flowchart will be described in the ordershown, no order of the steps should be necessarily implied. Steps of theflowcharts herein can be added, reordered, deleted, and combined withoutdeparting from the scope and spirit of the invention. For example, thedata receiving steps are shown first as may occur when chromatographicseparation data is read in from a storage device or network. However, ifthe data is processed in real-time, the data receiving steps may beinterlaced in the other steps (see FIG. 9) so no order should be impliedfrom the order in which the steps are shown.

At a step 181, the computer system receives a series of measurements fora first sample at a scanning location over time. The computer systemreceives a series of measurements for a second sample at a scanninglocation over time at a step 183. The series of measurements can beoptionally displayed as a plot of intensity vs. time.

The computer system displays the series of measurements for the firstsample as a series of bands at a step 185. As mentioned previously, theseries of bands resembles a conventional electrophoresis gel. At a step187, the computer system identifies one or more peaks in the series ofmeasurements for the first sample that corresponds to a marker. Ingeneral, peaks in the series of measurements can indicate the presenceof the labeled markers or constituents at the scanning location. At astep 189, the computer system identifies one or more peaks in the seriesof measurements for the second sample that corresponds to a marker. In apreferred embodiment, the peaks of markers are identified by a differentwavelength that is exhibited by the labels on the markers as compared tothe constituents.

At a step 191, the computer system scales the series of measurements forthe second sample so that the marker or markers have the samemeasurement. For multiple markers, a linear stretch or compression usinga point-to-point fit can be utilized. The computer system displays theseries of measurements for the second sample as a series of bands thatare aligned with and adjacent to the bands for the first sample at astep 193.

In order to illustrate the flowchart of FIG. 7, FIGS. 8A and 8B showscreen displays that illustrate the normalizing process. In FIG. 8A,each sample is processed serially and as they are processed, the seriesof measurements are shown as graphical plots of intensities vs. time inwindow area 167 and a series of bands in a window area 171. As shown,sample B2 is being processed. A series of bands 201 is being displayed,where the top and bottom bands correspond to markers. In preferredembodiments, the bands that correspond to markers are displayed in avisually different manner (e.g., a different color) so the user can morereadily identify the markers. However, it should be seen that series ofbands 201 does not align with a series of bands 203 for sample B1 thatwas previously processed. As the series of bands are not aligned, it maybe difficult to accurately compare the samples.

FIG. 8B shows the processing of the next sample, after the display ofthe data for sample B2 is normalized by the process shown in FIG. 7. Asshown, series of bands 201 is now aligned with series of bands 203 (andall the previously processed samples). As sample B3 is being processed,it can be seen from a series of bands 205 that corresponds to the samplethat it would also be beneficial to normalize series of bands 205.

Although FIGS. 8A and 8B show the series of bands being aligned to eachother, the series of bands can also be aligned to predeterminedlocations on the screen. For example, a single marker in each sample canbe utilized to align each displayed series of bands to a commonbaseline. Additionally, two markers in each sample can be utilized toalign each displayed series of bands to a displayed scale.

FIG. 9 shows a flowchart of another process of normalizingchromatographic separation data in which the samples include one or moremarkers. In general, the flowchart serially processes each sample untilall the samples have been processed. At a step 231, the computer systemreceives a series of measurements for a current sample at a scanninglocation over time. The series of measurements can be optionallydisplayed as a plot of intensity vs. time.

At a step 187, the computer system identifies one or more peaks in theseries of measurements for the current sample that corresponds to amarker. The computer system scales the series of measurements for thecurrent sample at a step 235. The series of measurements can be scaledso that the displayed bands that correspond to the marker or markers arealigned when displayed. Additionally, the series of measurements can bescaled to predetermined locations on the screen. The computer systemdisplays the series of measurements for the current sample as a seriesof bands that are aligned with and adjacent to the bands for a previoussample (if any) at a step 237. If it is determined that there are moresamples to process at a step 239, the flow returns to step 231.

FIGS. 10A-10E will illustrate some other innovative features ofembodiments of the invention. FIG. 10A shows a screen display where allthe sample wells have been processed. However, in processing two of thesamples, it was determined that they did not have the requisite numberof peaks (or the peaks did not satisfy certain criteria). Accordingly,series of bands 251 are shown with warning symbols that not enough peakswere detected. Additionally, a warning symbol 253 is displayed with atextual description of the warning since one of the samples withpotentially bad data, sample D3, is currently selected.

FIG. 10B shows a screen display in which the series of bands are shownin window area 167. Window 161 includes a toolbar 261. When a button 263is activated, the series of bands are displayed in window area 167.Additionally, the graphical plot of intensity vs. time for the currentlyselected sample, sample D3, is displayed in window area 171. It may beobserved that the series of bands in window area 167 are not normalized.A user can display the series of bands unaligned (i.e., as raw data) byactivating a button 265.

FIG. 10C shows a screen display where a single graphical plot is shownin window area 167. When a user activates a button 271, the graphicalplot of the currently selected sample is enlarged and displayed alone inwindow area 167. Numbers 273 are utilized to identify each peak inwindow area 167. The window area includes a data table 275 that showsdata for each of the numerically designated peaks. The data table shownincludes the migration time, area of the peak, and a signal to noiseratio, which can be calculated by dividing the peak height by the wellnoise. Additionally, the size of the macromolecule represented by thepeak (shown here in base pairs), concentration and molarity can beentered as properties of the assay and displayed in data table 275.Accordingly, the graphical plot of intensity vs. time can include thenumber of peaks and information regarding the data for each peak.

FIG. 10D shows a screen display where the display of the series of bandsis inverted. Button 263 has been activated to display the series ofbands in window area 167. As shown, the series of bands are normalizedfor easier comparison. A button 281 was activated that inverted thedisplay of the series of bands. A user may prefer to see the series ofbands inverted and activating button 281 will invert the display of theseries of bands to their previous state.

FIG. 10E shows a screen display where the user is able to modify thepeak find settings. A button 291 can be activated to display the peakfind settings so that the user may alter the way in which the data isanalyzed. When button 291 is activated, a window 293 appears that showsthe current peak find settings. The minimum peak height value determineswhether or not a peak is kept. For each peak, the difference between thebaseline and signal at the center point must be greater than the minimumpeak height value. The slope threshold setting determines the differencein the slope that must occur in order for a peak to begin. The inverseof this value is used to determine the peak end.

The first and last peak time settings determine the window in whichpeaks will be found. Any peaks outside these settings will be rejectedor ignored. The upper marker setting can be set to “nearest peak” or“last peak.” The “last peak” setting refers to the last peak kept afterthe peak find algorithm is finished. The “nearest peak” setting refersto the peak that falls nearest the upper marker in the ladder from thefirst (or other specified) well. In preferred embodiments the “lastpeak” setting is the default.

FIG. 11 shows a flowchart of a process of displaying chromatographicseparation data for multiple samples. As described above, the basicsteps performed in the display of chromatographic information accordingto the present invention can begin by acquiring this information using amicrofluidic instrument at a step 301. The output of the detectionsystem is a signal that varies with the fluorescence of the materialpassing through the detector at the time. The present invention not onlyprovides the ability to convert this serial stream of data into a moreconventional format, but also to display the serially acquired data in aparallel format.

The standards introduced into the samples are preferably such that theyare detected much earlier and much later than any of the constituentsthat might be expected to occur in the given sample, e.g., they havesmaller and/or larger molecular weights. Such standards would thus beexpected to occur before and after such constituents in a system such asthat described above. Alternatively, internal standards may be used,such that the standards occur interspersed within the range of expectedconstituents.

In addition to acquiring chromatographic data for the samples beinganalyzed, chromatographic data can be acquired for a standard “ladder”of molecular species having known characteristics (e.g., molecularweight, charge, or other characteristic) over a given time period. Thisstandard ladder can be used to generate a normalization curve, with thestandards creating a curve that relates migration time to the knowncharacteristic (e.g., molecular weight, charge, or the like) at a step303. Using this information, each set of bands for each sample may benormalized such that the sample in each lane displayed may be properlycompared to each of the other samples. This is done in the followingmanner.

At a step 305, the position of the markers in the given sample isdetermined. Next, fluorescence values are calculated for each positionin the display of the sample currently being displayed at a step 307. Itis at this point that the values of the unknown constituents are mappedto positions on the corresponding lane of the display. Thus, asmentioned above, the present invention converts the serial data into amore conventional parallel format. Normally, the sample data sodisplayed will then be normalized using the curve generated using thestandard ladder. At a step 309, the results for the current sample aredisplayed. Finally, at a step 311, the process is repeated if moresamples remain to be displayed.

FIG. 12 illustrates in further detail a flowchart of a preferred processof generating a graphical display of chromatographic data for onesample, according to the present invention. Again, the method begins byacquiring chromatographic data in some manner at a step 401. In thisembodiment, standards having extreme molecular weights (relative to thatof the sample's expected constituents) are introduced into the sample.The sample, along with the standards or markers therein, are run throughthe detection system. The smaller (i.e., lower molecular weight)fragments will normally be present at the output first (the smallerstandard being presented before all others, ideally), followed byincreasingly larger (i.e., greater molecular weight) fragments, followedat last by the larger of the two standards.

Next, at a step 403, the position of each of the standard markers isdetermined. This basically sets the range of possible values that willbe displayed, assuming that none of the sample's constituents are largeror smaller than the standards employed. At a step 405, the intensity ofthe standard marker is determined so that the intensity of each bandcreated by the sample's constituents may be scaled to a relative scale(arbitrary units are normally used in such a case).

At a step 407, the position of each of the constituents (as representedby one or more lines in the eventual displayed data) is scaled to therange determined in step 403. At a step 409, the intensity of eachconstituent is scaled to the arbitrary scaled just described. Thisinformation is then presented in a graphical format at a step 411.

FIGS. 13 and 14 illustrate a graphical display of chromatographic data(also referred to herein as a “gel display”) according to one embodimentof the present invention. FIG. 13 illustrates a gel display using themore conventional light-on-dark color scheme reminiscent of agarosenucleic acid slab gels stained with fluorescent dyes. However,embodiments of the present invention are capable of displaying the givenchromatographic data using any color scheme, allowing the user to adjustboth foreground and background colors to improve the visibility ofvarious features of the chromatographic data being displayed. Moreover,different bands (i.e., fragment sizes) may be displayed using differentcolors, allowing easy identification of the various constituents beingdisplayed. In some embodiments, a user can change the contrast,brightness or perform “gamma” correction to facilitate viewing the geldisplay.

For reasons of clarity, the display illustrated in FIG. 14 will bedescribed, although the following comments apply equally to FIG. 13. Astandard ladder 400 and samples 410, 420, 430, and 440 are displayed ina gel display window 450 in FIG. 14. Standard ladder 400 containsnumerous fragments of known size (i.e., standard-size fragments), whichare displayed as bands 451-461. Sample ladders 410, 420, 430, and 440also contain standard-size fragments corresponding to the fragmentsrepresented by bands 451 and 461. These are shown as bands 470, 472,474, and 476, and bands 471, 473, 475, and 477, respectively. Thesamples' constituents are shown as sets of bands 480-483. As can beseen, samples 410, 420, 430, and 440 are substantially similar. This isevident because the position, width, and other characteristics of thebands in each of sets of bands 480-483 are substantially similar.

As can be seen, the present invention matches the smallest and largeststandard fragments in each of sample ladders 410, 420, 430, and 440 tothose in standard ladder 400 (i.e., bands 451 and 461). The display iscalibrated using bands 452-460 of standard ladder 400. Thus, the sizeand position of one or more bands in sets of bands 480-483 may then bedetermined by determining the given band's position using, for example,a “rollover” feature. This feature allows a selected position on theinterpolation curve or on a lane of the gel display to be identifiedusing the screen cursor. This position may then be related to a givenmolecular weight, fragment length, or other criteria of the constituentsof the samples being analyzed. In this manner, the user can obtaininstantaneous display of the characteristic (molecular weight, fragmentlength, or the like) by simply placing the cursor over the band ofinterest. Alternatively, each band can be automatically identified, anda fragment size displayed by the band in question.

The present invention offers several advantages. For example, once thechromatographic data has been analyzed and converted into a gel format,several advantageous features may be provided. A major advantage of thepresent invention is the invention's ability to display data collectedserially in a more conventional format. Moreover, the present inventionpermits a single standard ladder to be analyzed once for any number ofruns, using the preferred chromatographic data collection system, forexample. In the prior art, a standard ladder must be run for each gel,because gel characteristics vary from gel to gel. Thus, a lane is usedin each and every gel that is run. In a preferred embodiment of thepresent invention, because there would be no substantial difference fromrun to run, only a single ladder would need to be run, saving time andlowering operating expenses.

In a further advantage, the data in the gel format is digitized, makingits display very flexible compared to conventional gels. For example,when displaying the analog of a protein gel, the gel display may use alight coloring on a dark background to emulate a silver halide process(normal contrast, as shown in FIG. 13), or a dark coloring on a lightbackground to emulate a lithium bromide process (reverse contrast, asshown in FIG. 14). Further, the digitized gel is easily stored, printed,and reproduced from its digitized format.

Another advantage is the ability to automatically align the variousconstituents represented in two or more samples to markers included inthe samples. This may be necessary if the raw data from various samplesdoes not match up properly, or is skewed for some reason (e.g., variedseparation conditions). For example, if two samples are to be compared,but the samples differ in the ranges of molecular weights of theconstituents therein (or fail to match up for some other reason), theirmarkers may be matched/aligned. Thus, one or both of the samples' gelrepresentations are translated from their current state to a translatedstate in which each point is mapped from its current position to a newposition. When this process is completed, each marker in the firstsample should substantially match each marker in the second sample. Thisprocess is referred to herein as warping. Internal markers may be usedin such a situation to further improve the accuracy of such warping.This warping allows for a display according to the present invention toaccount for non-linearities that may vary from sample to sample, whendisplaying such samples for comparison.

Finally, the graphical display of the present invention allows systemsthat generate data in a serial fashion to display and compare such datain parallel. In other words, for a system that records fluorescence datafor each sample on after the other, the present invention allows theviewing of such data as a parallel set of lanes. This is similar to atraditional gel, in which multiple lanes are generated. However, unlikethe traditional gel, the present invention is not forced to display thedata in this manner. In a traditional gel, the number of lanes usedshould be maximized because the gel cannot be reused. Because aprocessing system that generates data serially runs analyses one at atime and the present invention stores and displays that information at alater time, no such limitations are imposed. Thus, the present inventionmay display the chromatographic separation data singly, in pairs, or inany other configuration that the user finds advantageous.

The invention has now been explained with reference to specificembodiments. Other embodiments will be apparent to those of ordinaryskill in the art in view of the foregoing description. For example, theinvention can be advantageously applied to other microfluidic devicesand various types of molecules in addition to those described herein. Itis therefore not intended that this invention be limited except asindicated by the appended claims along with their full scope ofequivalents.

1. A computer implemented method of displaying chromatographicseparation data, comprising: displaying using a computer system a firstseries of measurements for a first sample as a first lane comprising afirst series of bands, wherein the first series of measurementsindicates the presence of constituents in the first sample, the firstsample constituents detected at a single point along the length of achannel as they move through the channel past that point; and displayingusing the computer system a second series of measurements for a secondsample as a second lane comprising a second series of bands, the secondsample constituents detected at the single point along the length of thechannel as they move through the channel past that point, wherein thefirst lane and the second lane are displayed as a parallel set of lanes.2. The computer-implemented method of claim 1, further comprising:receiving input to invert the display of the first and second series ofbands; and inverting the displayed first and second series of bands. 3.The computer-implemented method of claim 1, wherein the first and secondsamples include a marker and wherein each of the first and second seriesof measurements indicates presence of the marker, the marker detected atthe single point along the length of the channel as it moves through thechannel past that point.
 4. The computer-implemented method of claim 3,the method further comprising: identifying one or more bands in thefirst series of bands that correspond to the marker; identifying one ormore bands in the second series of bands that correspond to the marker;aligning the one or more bands in the first series of bands thatcorrespond to the marker and the one or more bands in the second seriesof bands that correspond to the marker with a predetermined location,thereby aligning the first and second series of bands to a commonbaseline
 5. The computer-implemented method of claim 3, the methodfurther comprising: displaying the one or more bands that correspond tothe marker in each sample visually different from the bands thatcorrespond to the constituents in each sample.
 6. Thecomputer-implemented method of claim 1, wherein the first series ofmeasurements and the second series of measurements comprise separationdata obtained from a process carried out on a microfluidic device.
 7. Asystem comprising: a device on which at least first and second samplesare subjected to a chromatographic separation process; an instrumentconfigured to receive the device, the instrument comprising a detectionsystem that detects results of the chromatographic separation processand instrumentation for controlling fluid or material transport anddirection within the device; and a computer system that displayschromatographic separation data corresponding to results received fromthe instrument, wherein the computer system comprises: a processor; anda non-transitory computer readable medium coupled to the processor thatstores a computer program that presents the chromatographic separationdata from each sample as a lane comprising a series of bands; whereinthe computer program presents a first lane comprising a series of bandsfrom chromatographic separation data from the first sample and a secondlane comprising a series of bands from chromatographic separation datafrom the second sample as a parallel set of lanes.
 8. The system ofclaim 7, wherein the computer program also identifies a band in thefirst series of bands that corresponds to a marker contained in thefirst sample, identifies a band in the second series of bands thatcorresponds to the same marker contained in the second sample, andaligns the bands when presenting the first and second lanes as aparallel set of lanes.